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Objectives SSc patients are recognized as presenting an increased risk of altered bone mass. The aim of this study was to assess the bone quality, by trabecular bone score (TBS), in SSc patients in correlation with different levels of microvascular damage, as evaluated by nailfold videocapillaroscopy (NVC), and to compare the results regarding bone quality with RA patients and healthy subjects (CNT). Methods Eighty-four SSc patients, 98 RA patients and 60 CNT, were studied. BMD (g/cm2) of the lumbar spine (L1–L4) was analysed by DXA scan. Lumbar spine bone quality was derived from each spine DXA examination using the TBS analysis. NVC patterns were analysed. Results A total of 56/84 SSc patients (66%) as well as 78/98 RA patients (80%) showed bone loss at DXA and BMD was found to be significantly lower than in the CNT (P < 0.001). Similarly, lumbar spine TBS was found to be significantly lower in SSc and RA patients than in CNT (P < 0.001). TBS values were found to be lower in SSc with a late NVC pattern, compared with the active or early pattern (late vs active and early pattern, P < 0.001). There was no statistically significant difference in the mean lumbar spine TBS between SSc and RA patients (P = 0.238). Conclusion The data obtained showed significantly lower bone quality (lower TBS and BMD) in SSc and RA patients compared with CNT. The bone quality seemed lower in SSc patients with more altered microvasculature (late NVC pattern). [ABSTRACT FROM AUTHOR]

Objectives. Increased release of neutrophil extracellular traps (NETs) is implicated in the activation of plasmacytoid dendritic cells, vascular disease and thrombosis in SLE and APS. However, studies comparing NET release between patients with SLE and APS are lacking. Here we evaluated plasma-induced NET release in a large cohort of patients with SLE, SLE + APS and primary APS in relation to clinical and serological parameters. Methods. Neutrophils from healthy controls were exposed to plasma of heterologous healthy controls (n = 27) or SLE (n = 55), SLE + APS (n = 38) or primary APS (PAPS) (n = 28) patients and NET release was quantified by immunofluorescence. In a subset of SLE patients, NET release was assessed in longitudinal samples before and after a change in treatment. Results. Plasma-induced NET release was increased in SLE and APS patients, with the highest NET release found in patients with SLE (±APS). Plasma of 60% of SLE, 61% of SLE + APS and 45% of PAPS patients induced NET release. NET release did not correlate with disease activity in SLE or APS. However, increased levels of anti-nuclear and anti-dsDNA autoantibodies were associated with increased NET release in SLE and APS. Only in SLE patients, elevated NET release and an increased number of low- density granulocytes were associated with a high IFN signature. Conclusion. Increased NET release is associated with autoimmunity and inflammation in SLE and APS. Inhibition of NET release thus could be of potential benefit in a subset of patients with SLE and APS. [ABSTRACT FROM AUTHOR]

Objectives. SSc is a devastating disease that results in fibrosis of the skin and other organs. Fibroblasts are a key driver of the fibrotic process through deposition of extracellular matrix. The mechanisms by which fibroblasts are induced to become pro-fibrotic remain unclear. Thus, we examined the ability of SSc keratinocytes to promote fibroblast activation and the source of this effect. Methods. Keratinocytes were isolated from skin biopsies of 9 lcSSc, 10 dcSSc and 13 control patients. Conditioned media was saved from the cultures. Normal fresh primary fibroblasts were exposed to healthy control and SSc keratinocyte conditioned media in the presence or absence of neutralizing antibodies for TGF-β. Gene expression was assessed by microarrays and real-time PCR. Immunocytochemistry was performed for α-smooth muscle actin (a-SMA), collagen type 1 (COL1A1) and CCL5 expression. Results. SSc keratinocyte conditioned media promoted fibroblast activation, characterized by increased α-SMA and COL1A1 mRNA and protein expression. This effect was independent of TGF-β. Microarray analysis identified upregulation of nuclear factor κB (NF-κB) and downregulation of peroxisome proliferator-activated receptor γ (PPAR-γ) pathways in both SSc subtypes. Scleroderma keratinocytes exhibited increased expression of NF-κB-regulated cytokines and chemokines and lesional skin staining confirmed upregulation of CCL5 in basal keratinocytes. Conclusion. Scleroderma keratinocytes promote the activation of fibroblasts in a TGF-β-independent manner and demonstrate an imbalance in NF-κB1 and PPAR-γ expression leading to increased cytokine and CCL5 production. Further study of keratinocyte mediators of fibrosis, including CCL5, may provide novel targets for skin fibrosis therapy. [ABSTRACT FROM AUTHOR]

Objective. There is growing understanding of the importance of bone in OA. Our aim was to determine the relationship between 3D MRI bone shape and total knee replacement (TKR). Methods. A nested case-control study within the Osteoarthritis Initiative cohort identified case knees with confirmed TKR for OA and controls that were matched using propensity scores. Active appearance modelling quantification of the bone shape of all knee bones identified vectors between knees having or not having OA. Vectors were scaled such that -1 and +1 represented the mean non-OA and mean OA shapes. Results. Compared to controls (n = 310), TKR cases (n = 310) had a more positive mean baseline 3D bone shape vector, indicating more advanced structural OA, for the femur [mean 0.98 vs -0.11; difference (95% CI) 1.10 (0.88, 1.31)], tibia [mean 0.86 vs -0.07; difference (95% CI) 0.94 (0.72, 1.16)] and patella [mean 0.95 vs 0.03; difference (95% CI) 0.92 (0.65, 1.20)]. Odds ratios (95% CI) for TKR per normalized unit of 3D bone shape vector for the femur, tibia and patella were: 1.85 (1.59, 2.16), 1.64 (1.42, 1.89) and 1.36 (1.22, 1.50), respectively, all P < 0.001. After including Kellgren--Lawrence grade in a multivariable analysis, only the femur 3D shape vector remained significantly associated with TKR [odds ratio 1.24 (1.02, 1.51)]. Conclusion. 3D bone shape was associated with the endpoint of this study, TKR, with femoral shape being most associated. This study contributes to the validation of quantitative MRI bone biomarkers for OA structure-modification trials. [ABSTRACT FROM AUTHOR]

Objectives. Conflicting reports of the effect of physical activity on knee cartilage may be due to the heterogeneity of populations examined and, in particular, the underlying health of the knee joint. This study examined the influence of recreational and occupational physical activity on cartilage volume loss. Methods. A total of 250 participants with no significant musculoskeletal disease were recruited. A gender-specific median cartilage volume split was used to define people in the lowest and highest 50% of baseline cartilage volume. Baseline recreational and occupational activity was examined by questionnaire, while cartilage volume was assessed by MRI at baseline and 2.4 years later. Results. Significant interactions were demonstrable between physical activity and cartilage volume loss based on stratification of baseline cartilage volume (all P ≤ 0.03). There was a dose-response relationship between frequently performed baseline occupational activities and medial cartilage volume loss in both the low (B = 0.2% per annum, 95% CI: 0.0, 0.04% per annum) and high (B = -0.2% per annum, 95% CI: -0.4, 0.0% per annum) baseline cartilage volume groups (P = 0.001 for interaction). Individuals with low baseline cartilage volume who were active in their occupation and/or recreational activity had greater medial cartilage volume loss than their more inactive counterparts (2.4% per annum vs 1.5% per annum, P = 0.02). Conclusion. Whereas people with less baseline cartilage volume are more at risk of structural knee damage with either heavy occupational or recreational workloads or both, individuals with high baseline cartilage volume may advantageously modify their risk for knee OA by participating in more frequent occupational physical activities. [ABSTRACT FROM AUTHOR]

Objective. The aim of the study was to assess the association between plethysmographically measured vasospasms during stepwise cooling and recovery, as an index for digital ischaemia, and nailfold capillaroscopic pattern (NCP) severity in patients with primary or secondary RP, including SSc. Methods. In 381 consecutive patients with suspected RP without a history of digital ulcers, NCP (assessed by widefield videocapillaroscopy), fingertip photoelectric plethysmography during cooling and recovery and clinical characteristics were analysed. NCPs were graded as follows: normal, non-specific, early and active. The mean ischaemic time was defined as the mean time of perfusion loss during cooling and recovery of five fingers. Results. In the patients with loss of perfusion during cooling and recovery, the NCP was normal in 152, non-specific in 96, early in 61 and active in 39 patients. The mean ischaemic time was positively associated with the severity of NCP, with P < 0.05 for each two- or three-grade increase and independent of underlying SSc. The difference was most pronounced during recovery. Conclusion. We demonstrate that the degree of vasospasm and ischaemia provoked by stepwise cooling and recovery are positively associated with NCP in patients with RP of different aetiologies and without a history of digital ulcers. [ABSTRACT FROM AUTHOR]

Objective. IL-9 has been shown to be upregulated before the clinical onset of articular disease in RA. The exact role of IL-9 and Th9 cells in RA, however, has not yet been adequately studied. The aim of this study was to evaluate the expression of IL-9 and IL-9-expressing cells in RA patients. Methods. IL-9, IL-9R, PU.1, IL-9, thymic stromal lymphopoietin (TSLP), IL-4 and TGF-α expression was assessed by real-time-PCR in the synovial tissues of RA and OA patients. IL-9, IL-9R, IL-4, TSLP and TGF-β were also investigated by immunohistochemistry. Peripheral CD4+ T cell subsets were studied by flow cytometry analysis before and after incubation with citrullinated peptides. Results. IL-9 was overexpressed in RA synovial tissues and correlated with the degree of histological organization of B and T cells in ectopic lymphoid structures. The majority of IL-9-producing cells were identified as CD3+ cells. Increased mRNA and protein expression of IL-9R, IL-4, TSLP and TGF-β was also observed in RA synovial tissue. Blood peripheral Th9 cells were expanded by citrullinated peptides. Conclusion. These results indicate that Th9 cells and IL-9 were frequently detected in peripheral blood mononuclear cells and synovia of RA patients. A possible pathogenic role for Th9 in RA is discussed. [ABSTRACT FROM AUTHOR]

Synovial fibrosis is often found in OA, contributing heavily to joint pain and joint stiffness, the main symptoms of OA. At this moment the underlying mechanism of OA-related synovial fibrosis is not known and there is no cure available. In this review we discuss factors that have been reported to be involved in synovial fibrosis. The aim of the study was to gain insight into how these factors contribute to the fibrotic process and to determine the best targets for therapy in synovial fibrosis. In this regard, the following factors are discussed: TGF-β, connective tissue growth factor, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2, tissue inhibitor of metalloproteinase 1, A disintegrin and metalloproteinase domain 12, urotensin-II, prostaglandin F2α and hyaluronan. [ABSTRACT FROM AUTHOR]

Objective. Diseases associated with calcium-containing crystal deposition can lead to local bone erosion. We aimed to determine whether calcium-containing crystal-hydroxyapatite, β-tricalcium phosphate and CPPD enhanced osteoclastogenesis and to define underlying mechanisms of action. Methods. Osteoclastogenesis was studied by culturing murine RAW 264.7 osteoclast precursor cells with RANK ligand (RANKL)/ M-CSF and/or calcium-containing crystals, and observing the tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and TRAP activity. Resorption pit formation was used to evaluate osteoclast activity. Real-time RT-PCR analysis revealed osteoclast marker genes, including TRAP, cathepsin K and calcitonin receptor (CTR). Western blotting was used to analyse the phos-phorylation levels of signal transduction molecules. Results. Three kinds of calcium-containing crystal significantly enhanced RANKL/M-CSF-induced osteoclastogenesis in RAW 264.7 cells, as evidenced by the increased number of TRAP-positive multinucleated cells, TRAP activity and resorption pit formation in a dose-dependent manner. Hydroxyapatite, β-tricalcium phosphate and CPPD treatments significantly enhanced RANKL/M-CSF-induced mRNA expression of TRAP, cathepsin K and CTR. Moreover, the three kinds of calcium-containing crystal enhanced the phosphorylation of extracellular-signal-regulated kinase and p38 in RANKL/M-CSF-treated cells. Conclusion. We concluded that calcium-containing crystals can promote osteoclastogenesis and bone resorption through the extracellular-signal-regulated kinase and p38 pathways. Together with synovial activation, this mechanism may be important in the pathogenesis of destructive arthropathies triggered by calcium-containing crystals. [ABSTRACT FROM AUTHOR]

Objective. To determine the prevalence of asymptomatic cardiac involvement and its correlation with non-cardiac manifestation in Thai SSc patients. Methods. A cross-sectional study was carried out between January 2012 and June 2013 at Srinagarind Hospital, Khon Kaen University, Thailand, on adult SSc patients without signs or symptoms suggestive of cardiac involvement. We excluded those with overlap syndrome, having serum creatinine >123.8 µmol/l, history of cardiac diseases, any atherosclerosis risk factors and receiving angiotensin-converting enzyme inhibitors. Non-invasive tests related to cardiac involvement were performed, including: echocardiography, ECG, chest X-ray, inflammatory biomarkers, cardiac enzymes and N-terminal prohormone of brain natri-uretic peptide. Results. A total of 103 SSc patients were enrolled, 61.2% of whom were in the subset. Of these, 63 patients had at least one test abnormality (prevalence 61.2%; 95% CI 51.6, 70.7). The two leading cardiac abnormalities were diastolic dysfunction (44.7%) and elevation of cardiac enzymes (36.9%). The only predictor for cardiac involvement per multivariate analysis was the dcSSc subtype with a higher modified Rodnan skin score, and shorter disease duration (odds ratio = 3.37; 95% CI 1.07, 10.65). Compared with the limited subtype, dcSSc was also significantly associated with elevated cardiac enzyme and prolonged distance between a Q wave and T wave in an ECG (QT interval). Conclusion. Asymptomatic cardiac involvement in Thai SSc was not uncommon, and the most common finding was diastolic dysfunction. Elevated cardiac enzymes were found in one-third of the patients, which correlated with the dcSSc subtype with a higher modified Rodnan skin score and shorter disease duration, suggestive of early myocardial microcirculation disruption. Long-term follow-up was performed to elucidate the clinical implication of these abnormalities. [ABSTRACT FROM AUTHOR]

Objective. GCA is a large- and medium-vessel arteritis characterized by a range of histological patterns of vascular wall injury. The aim of this study was to immunologically characterize the various histological patterns of GCA. Methods. Thirty-five consecutive patients with biopsy-proven GCA and 15 normal controls were studied. IL-8, IL-9, IL-9R, IL-17, IL-4, TGF-β and thymic stromal lymphopoietin expression was evaluated by RT-PCR and immunohistochemistry on artery biopsy specimens. Confocal microscopy was used to characterize the phenotypes of IL-9-producing and IL-9R-expressing cells. Five additional patients who had received prednisone when the temporal artery biopsy was performed were also enrolled to evaluate the effect of glucocorticoids on IL-9 and IL-17 expression. Results. IL-17 overexpression was observed mainly in arteries with transmural inflammation and vasa vasorum vasculitis. IL-9 overexpression and Th9 polarization predominated in arteries with transmural inflammation and small-vessel vasculitis. The tissue expression of both IL-9 and IL-17 was correlated with the intensity of the systemic inflammatory response. IL-4, TGF-β and thymic stromal lymphopoietin, which are involved in the differentiation of Th9 cells, were overexpressed in arteries with transmural inflammation and small-vessel vasculitis. IL-9R was also overexpressed in GCA arteries with transmural inflammation and was accompanied by increased expression of IL-8. Conclusion. Herein we provide the first evidence that distinct populations of potentially autoreactive T cells, expressing different cytokines (Th17 vs Th9), characterize patients with particular histological subsets of GCA and may thus contribute to the heterogeneity of tissue lesions observed in these patients. [ABSTRACT FROM AUTHOR]

Objective. B cells play an important role in the pathogenesis of autoimmune diseases. The role of Bruton's tyrosine kinase (Btk) in cytokine-induced human B cell differentiation and class-switch recombination remains incompletely defined. This study analysed the effect of Btk on human activated B cells. Methods. Purified B cells from healthy subjects were stimulated with B cell receptor (BCR) and other stimuli with or without a Btk inhibitor and gene expression was measured. The B cell line BJAB was used to assess Btk-associated signalling cascades. Phosphorylated Btk (p-Btk) in peripheral blood B cells obtained from 10 healthy subjects and 41 patients with RA was measured by flow cytometry and compared with patient backgrounds. Results. IL-21 signalling, in concert with BCR, CD40 and BAFF signals, led to robust expression of differentiation- and class-switch DNA recombination-related genes and IgG production in human B cells, all of which were significantly suppressed by the Btk inhibitor. Although phosphorylation of STAT1 and STAT3 was induced by co-stimulation with IL-21, BCR and CD40, STAT1 phosphorylation in the nucleus, but not in the cytoplasm, was exclusively impaired by Btk blockade. High levels of p-Btk were noted in B cells of RA patients compared with controls and they correlated significantly with titres of RF among RF-positive patients. Conclusion. The findings elucidate a model in which Btk not only plays a fundamental role in the regulation of BCR signalling, but may also mediate crosstalk with cytokine signalling pathways through regulation of IL-21-induced phosphorylation of STAT1 in the nuclei of human B cells. Btk appears to have pathological relevance in RA. [ABSTRACT FROM AUTHOR]

Objective. IL-35 is the most recently identified member of the IL-12 family. It consists of EBV-induced gene 3 (EBI3) and IL-12α chain p35. We investigated whether IL-35 enhances the in vitro immunosup-pressive function of peripheral blood isolated from patients with RA. Methods. Peripheral blood was harvested from 17 active and 10 inactive RA patients and IL-35 concentrations were quantified using an ELISA. An expression vector containing IL-35 with a FLAG tag at the carboxyl-terminus was constructed by covalently linking EBI3 and IL-12α (p35). The function of IL-35 was then evaluated in a suppression assay using T cells isolated from human RA patients with CD2, CD3 and CD28 antibodies. Results. Serum IL-35 levels and the number of Treg were decreased significantly in patients with active RA. There was a significant correlation between serum IL-35 and the 28-joint DAS with ESR (DAS28-ESR) in patients with active RA. IL-35 treatment enhanced the regulatory function, suppressing the levels of inflammatory cytokines such as IL-17 and IFN-γ and the cellular growth of effector T cells stimulated by conjugation with CD2, CD3 and CD28. Conclusion. These data revealed that IL-35 might suppress T cell activation during the peripheral immune responses of RA. Therefore our data suggest that IL-35 might have multiple therapeutic targets. [ABSTRACT FROM AUTHOR]

Objective. Proline-serine-threonine-phosphatase-interacting protein 2 (PSTPIP2) is involved in macrophage activation, neutrophil motility and osteoclast differentiation. However, the role of PSTPIP2 in inflammation and autoinflammatory diseases is still not clear. In this study, we generated PSTPIP2 knockout (Pstpip2-/-) mice to investigate its phenotype and role in autoinflammatory diseases. Methods. We constructed a Pstpip2-targeting vector and generated Pstpip2-/- mice. The phenotype and immunopathology of Pstpip2-/- mice were analysed. Results. All Pstpip2-/- mice developed paw swelling, synovitis, hyperostosis and osteitis, resembling SAPHO syndrome, an inflammatory disorder of the bone, skin and joints. Multifocal osteomyelitis was found in inflamed paws, with increased macrophage and marked neutrophil infiltrations in the bone, joint and skin. Profound osteolytic lesions with markedly decreased bone volume density developed in paws and limbs. Neutrophil-attracting chemokines and IL-1b were markedly elevated in inflamed tissues. Conclusion. Our study suggests that PSTPIP2 could play a role in innate immunity and development of autoinflammatory bone disorders, and may be associated with the pathogenesis of human SAPHO syndrome. [ABSTRACT FROM AUTHOR]

Objectives. Chemerin is a member of adipocytokines with a chemoattractant effect on plasmacytoid dendritic cells and macrophages and pro-angiogenic properties. We investigated the potential role of chemerin in the development of SSc. Methods. Chemerin expression was evaluated by immunostaining and/or real-time quantitative RT-PCR in human and murine skin. The mechanisms regulating chemerin expression in dermal fibroblasts and endothelial cells were examined using the gene silencing technique and chromatin immunoprecipitation. Serum chemerin levels were determined by ELISA in 64 SSc patients and 19 healthy subjects. Results. In SSc lesional skin, chemerin was up-regulated in small blood vessels, while it was downregulated in fibroblasts surrounded with thickened collagen bundles. The decreased expression of chemerin was significantly reversed by TGF-b1 antisense oligonucleotide in cultured SSc dermal fibroblasts and chemerin expression was markedly decreased in dermal fibroblasts of bleomycin-treated mice. Gene silencing of transcription factor Fli1, which binds to the chemerin promoter, induced chemerin expression in human dermal microvascular endothelial cells and Fli1+/- mice exhibited elevated chemerin expression in dermal blood vessels. Serum chemerin levels inversely correlated with estimated glomerular filtration rate in SSc patients with renal dysfunction. In SSc patients with normal renal function, patients with digital ulcers had higher serum chemerin levels than those without. Conclusion. Chemerin is down-regulated in SSc dermal fibroblasts by autocrine TGF-b, while it is upregulated in SSc dermal blood vessels through endothelial Fli1 deficiency. Increased chemerin expression in dermal blood vessels may be associated with the development of digital ulcers in SSc. [ABSTRACT FROM AUTHOR]

Objective. Many cytokines involved in RA activate the Janus kinase–signal transducer and activator of transcription (JAK-STAT) pathways. Therapeutic drugs that inhibit these pathways are being developed for RA. To investigate disease-related alterations in the activity of JAK-STAT pathways in RA, we studied the expression and activation of STAT1 and STAT3 in unstimulated and cytokine-stimulated cells and determined the levels of circulating cytokines. Methods. The expression of STAT1 and STAT3 mRNA in peripheral blood (PB) and SF T cells and monocytes was studied in RA patients and healthy volunteers by RT-PCR. Basal and cytokine (IFN-γ, IL-6, IL-10)-induced STAT phosphorylation was analysed in PB T cells and monocytes using multicolour flow cytometric analysis. Results. STAT3 mRNA levels were up-regulated in both PB and SF T cells and monocytes from RA patients. STAT1 expression was elevated in SF monocytes. The levels of phospho-STAT3 in resting PB T cells and monocytes were significantly higher in patients with RA than in healthy volunteers. IL-6 levels were elevated in RA plasma and correlated with the level of STAT3 phosphorylation in CD4+ T cells and monocytes. IL-6-mediated STAT3 activation was deregulated in T cells from RA patients. IL-6-induced phosphorylation of STAT3 was decreased in CD4+ T cells from patients with high plasma IL-6 levels and constitutive STAT3 phosphorylation. Conclusion. The results suggest that IL-6 induces hyperactivation of STAT3 in circulating immune cells in active RA, and this subsequently desensitizes the IL-6 response in T cells. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Treg) are functionally defective in patients with RA. Restoring their function may not only control inflammation but also restore tolerance in these patients. Biologic therapies have been tremendously successful in treating RA. Here we review numerous reports suggesting that these immunomodulatory therapies have an impact on Treg and that this may contribute to their beneficial effects. Better understanding of their mode of action may not only lead to improvements in therapies and sustained remission but also enable the development of biomarkers of response, which would be the first steps towards personalized medicine. [ABSTRACT FROM AUTHOR]

Objective. The aim of this study was to investigate the turnover of Treg cells in the SF of RA patients. Methods. Treg cells were enumerated in peripheral blood and SF of RA patients and analysed by flow cytometry for expression of the proliferation marker Ki-67 and binding of the apoptosis marker annexin V. Sorted Treg cells of RA patients were analysed for expression of anti-apoptotic regulators Bcl-2 and microRNA-21 (miR-21) by RT-PCR. Results. Treg cells displaying a memory phenotype were abundant in the SF of RA patients. SF Treg cells more frequently expressed the proliferation marker Ki-67 than conventional T cells. Only few SF Treg cells were apoptotic, as indicated by limited annexin V staining of these cells. SF Treg cells displayed high transcription levels of Bcl-2 and miR-21 in comparison with SF conventional T cells and peripheral blood Treg cells. Conclusion. Treg cells with a memory phenotype accumulate in the SF of RA patients. These Treg cells have a high proliferative activity and demonstrate little apoptosis. The latter finding could be explained by high transcription of Bcl-2 and miR-21 in SF Treg cells. [ABSTRACT FROM AUTHOR]

Objective. SS is an autoimmune exocrinopathy affecting ~1 million patients in the USA that is diagnosed mostly in middle-aged women. Oral fluids (OFs) serving as the mirror of the body were suggested as an ideal non-invasive diagnostic tool. Previously we developed depletion techniques for OF high-abundance proteins to increase visualization of low-abundance proteins. Therefore the aim of this study was to examine the effect of depletion pretreatments on the identification potential of SS OF biomarker candidates. Methods. Unstimulated OFs were collected from 18 female SS patients and 18 healthy age- and gender-matched controls. High-abundance proteins were depleted using affinity and immunodepletion methodologies followed by semi-quantitative two-dimensional gel electrophoresis and quantitative dimethylation liquid chromatography tandem mass spectrometry (LC-MS/MS). To initially validate the MS results, western blotting was performed. Results. The use of depletion strategy before proteomics analysis increased identification ability by 3-fold. Overall, 79 biomarker candidates were identified. Proteins with the most pronounced fold changes were related to SS serum or tissue factors. In addition, bioinformatics analysis of proteins with a >3-fold increase in SS patients showed calcium-binding proteins, defence-response proteins, proteins involved in apoptotic regulation, stress-response proteins and cell motion-related proteins. Preliminary validation by western blotting of profilin and CA-I indicated similar expression profile trends to those identified by quantitative MS. Conclusion. The significance of OF novel depletion methodologies is clearly demonstrated for increased visibility of biomarker candidates as well as for unveiling possible mechanisms involved in this syndrome. This represents a major contribution to our ability to use OF as a future diagnostic fluid. [ABSTRACT FROM AUTHOR]

Objective. Increased numbers of IL-17-producing CD4+ T cells have been observed in AS. However, it is not known whether these CD4+ T cells are already present in early disease or if this is a late disease phenomenon only. Therefore we aimed to investigate whether IL-17-producing CD4+ T cells are involved in early active axial SpA, including patients without imaging abnormalities, by determining the frequency and phenotype of IL-17-producing CD4+ T cells in these patients. Methods. Flow cytometry was used to analyse cytokine production and surface marker expression of peripheral blood mononuclear cells from 31 patients suffering from early active HLA-B27-positive axial SpA fulfilling the Assessment of SpondyloArthritis International Society criteria with or without MRI abnormalities and 21 healthy controls. Results. Patients with early active axial SpA showed an increased percentage of IL-17-producing CD4+ T cells compared with healthy controls (mean 1.1% vs 0.4%, respectively; P = 0.013). The percentage of IL-17-producing CD4+ T cells was equally increased in patients with and without MRI abnormalities (1.2% vs 1.1%, respectively; P = 0.81). These IL-17-producing CD4+T cells expressed the αβT cell receptor but not the γδ T cell receptor, exhibited a memory phenotype and expressed CD161, but only sporadically expressed killer cell immunoglobulin-like receptor 3DL2 (KIR3DL2). Conclusion. IL-17-producing CD4+ T cells are increased in patients with early active axial SpA both with and without MRI abnormalities. This finding shows that the frequency of IL-17-producing CD4+ T cells is enhanced in the early stages of disease. [ABSTRACT FROM AUTHOR]

Objectives. We aimed to assess the effect of tocilizumab (TCZ), an IL-6 receptor inhibitor, on B, T, NK and NKT cells in patients with RA and to study the cell type predictors of remission. We also compared NK cells in patients with RA and in controls. Methods. RA patients included in the study met the 2010 ACR/European League Against Rheumatism (EULAR) criteria, were receiving stable doses of steroids and had not received rituximab in the previous year. Different B and T cell subsets, NK cells and NKT cells were assessed by flow cytometry along with perforin A and granzyme B to estimate NK cell cytotoxicity. Results. We included 92 RA patients, including 20 requiring TCZ treatment and 15 requiring anti-TNF drugs, and 25 controls. At baseline, the proportion of CD56dimCD16+CD3- NK cells was inversely correlated with the 28-joint DAS (DAS28). In TCZ-treated patients, the baseline proportion of CD3_CD56+ NK cells was inversely correlated with the change in DAS28 at 3 months and the proportion was 3-fold greater for patients with DAS28 remission at 3 months than other patients. Change in the proportion of CD56briCD16- NK cells was linearly correlated with change in the DAS28 at 3 months. The baseline proportion of NK cells did not predict change in disease activity at 3 months with anti-TNF therapy. The perforin content in NK cells increased with TCZ treatment. Conclusion. This study supports NK cell involvement in RA and in the TCZ mechanism of action. NK cells at baseline could be a predictive factor of TCZ response if results are confirmed. [ABSTRACT FROM AUTHOR]

Objective. CD200/CD200R1 signalling has an immunoregulatory effect on the activation threshold of the inflammatory immune response and maintains immune homeostasis. In this study we evaluated the status of CD200/CD200R1 interaction in patients with RA. Methods. The expression of CD200 and CD200R1 was examined by immunohistochemistry and flow cytometry and was compared between RA patients and healthy controls (HCs). Sorted CD4+ T cells were stained with carboxyfluorescein succinimidyl ester (CFSE) and annexin V-propidium iodide to evaluate the effect of CD200 on cell proliferation and apoptosis. The effect of CD200 on Th17 differentiation, function and osteoclastogenesis was determined by flow cytometry, transwell migration assay and immunocytochemistry, respectively. Results. The proportion of CD200+ cells and CD200R1+ cells in peripheral blood mononuclear cells, peripheral CD14+ cells and CD4+ T cells was significantly lower in the RA patients than in HCs, whereas the number of CD200+ cells was higher in synovium from RA patients than in that from HCs. After treatment with infliximab and MTX we found increased expression of peripheral CD200/CD200R1 that correlated with a decrease in the 28-joint DAS. CD200Fc in vitro partially inhibited CD4+ T cell proliferation, promoted CD4+ T cell apoptosis, reduced CD4+ T cell differentiation into Th17 cells and down-regulated CCR6-mediated Th17 chemotaxis in cells from RA patients. In addition, the engagement of the CD200 receptors on CD14+ cells with CD200Fc in vitro reduced osteoclastogenesis and inhibited CD14+ celldriven Th17 differentiation. Conclusion. Abnormal CD200/CD200R1 expression in RA may contribute to abnormal Th17 cell differentiation, chemotaxis and osteoclastogenesis. [ABSTRACT FROM AUTHOR]

The theory of myofascial pain syndrome (MPS) caused by trigger points (TrPs) seeks to explain the phenomena of muscle pain and tenderness in the absence of evidence for local nociception. Although it lacks external validity, many practitioners have uncritically accepted the diagnosis of MPS and its system of treatment. Furthermore, rheumatologists have implicated TrPs in the pathogenesis of chronic widespread pain (FM syndrome). We have critically examined the evidence for the existence of myofascial TrPs as putative pathological entities and for the vicious cycles that are said to maintain them. We find that both are inventions that have no scientific basis, whether from experimental approaches that interrogate the suspect tissue or empirical approaches that assess the outcome of treatments predicated on presumed pathology. Therefore, the theory of MPS caused by TrPs has been refuted. This is not to deny the existence of the clinical phenomena themselves, for which scientifically sound and logically plausible explanations based on known neurophysiological phenomena can be advanced. [ABSTRACT FROM PUBLISHER]

SS is a chronic systemic autoimmune disease characterized by decreased exocrine gland function. A variety of other disease manifestations may also be present, including general constitutional symptoms and extraglandular features. A multidisciplinary approach focused on both local and systemic medical therapies is needed as the disease has a wide clinical spectrum. The current treatment for SS is mainly symptomatic. However, there is evidence that systemic drugs are effective in controlling extraglandular manifestations of the disease. Overall evidence for the role of conventional immunosuppressive therapy is limited. A number of attempts to use biologic therapies have led to variable results. Biologic agents targeting B cells, such as rituximab, epratuzumab and belimumab, have shown promising results, but further studies are needed to validate the findings. Early-phase studies with abatacept and alefacept proved that T cell stimulation inhibition is another potentially effective target for SS treatment. Modulation or inhibition of other targets such as IFN, IL-6 and Toll-like receptor are also currently being investigated. We have summarized the available evidence regarding the efficacy of biologic treatments and discuss other potential therapies targeting pathways or molecules recognized as being involved in the pathogenesis of SS. [ABSTRACT FROM PUBLISHER]

Mesenchymal stem cells (MSCs) are multipotent cells with the capacity to undergo chondrogenic differentiation. Systemically administered MSCs have been shown to preferentially accumulate at sites of tissue damage and inflammation, thus MSC-based therapy holds great promise for the treatment of inflammatory diseases such as RA. Modulation of MSC homing may allow targeted delivery of systemically administered MSCs to damaged articular cartilage, where they can suppress immune-mediated cartilage destruction and contribute to cartilage repair via a combination of chondrogenic differentiation and paracrine stimulation of intrinsic residual repair. To harness the potential of MSC homing, a thorough understanding of the mechanism is key. This review discusses current knowledge of the mechanism of MSC homing to injured/inflamed tissue and its implications for targeted MSC-based therapy in arthritis. [ABSTRACT FROM PUBLISHER]

Objective. The aim of this study was to investigate the effect of umbilical cord mesenchymal stem cells (UC-MSCs) on circulating T follicular helper (cTfh) cells in primary SS (pSS) patients.Methods. The percentage of CXCR5+PD-1+CD4+ T cells in peripheral blood mononuclear cells (PBMCs) was analysed by flow cytometry. PBMCs were co-cultured with UC-MSCs by cell-to-cell contact or in a trans-well system. Naive CD4+ T cells were isolated from PBMCs and then co-cultured with UC-MSCs under Tfh cell-polarizing conditions. The percentage of CXCR5+PD-1+CD4+ T cells, carboxyfluorescein succinimidyl ester (CFSE) fluorescence intensity and annexin V were determined by flow cytometric analysis. Real-time PCR and Luminex cytokine assay were performed to detect mRNA expression and supernatant protein levels. The activity of indoleamine 2,3-dioxygenase (IDO) was measured by HPLC.Results. Increased frequency of cTfh cells was found in pSS patients and was positively correlated with serum anti-La/SSB levels and the European League Against Rheumatism SS Disease Activity Index score. In vitro, UC-MSCs suppressed the differentiation and proliferation of cTfh cells. Real-time PCR analysis showed significantly higher IDO mRNA expression on UC-MSCs when co-cultured with naive CD4+ T cells under Tfh cell-polarizing conditions in pSS patients. However, IDO mRNA expression on UC-MSCs was only a little higher when UC-MSCs were co-cultured with naive CD4+ T cells in a trans-well system. In addition, HPLC showed increased IDO enzymic activity in the supernatant of UC-MSCs co-cultured with naive CD4+ T cells under Tfh cell-polarizing conditions in pSS. The addition of the IDO inhibitor 1-MT partly reversed the suppressive effect of UC-MSCs on the differentiation of cTfh cells.Conclusion. These results suggest an inhibitory effect of UC-MSCs on the differentiation of cTfh cells via the secretion of IDO, and soluble factors secreted by activated CD4+ T cells might contribute to IDO secretion by UC-MSCs. [ABSTRACT FROM PUBLISHER]

Objectives. The aim of the study was to examine the effect of bosentan on blood flow in the hand in a subset of patients who had reduced blood flow relative to healthy subjects. Additionally, the relationship between blood flow in the hands of SSc patients and the presence of digital ulcers (DUs) was assessed.Methods. SSc patients with a recent history of DUs and healthy subjects were included. Patients were classified into four subgroups: no current DUs or pitting scars, pitting scars only, new DUs or persistent DUs. The hand was categorized into three regions of interest (ROIs) and blood flow was measured by laser Doppler perfusion imaging at baseline, 4 and 12 weeks. Patients who had a reduction in blood flow of >50% relative to healthy control subjects in ROI 1 on baseline were treated with bosentan for 12 weeks.Results. Fifty-two SSc patients and 51 healthy subjects were included in the analysis. There was no significant difference in blood flow in the hand across the patient subgroups at baseline. Sixteen SSc patients had a reduction of blood flow of ≥50% vs healthy subjects and received bosentan. Bosentan significantly (P < 0.05) increased blood flow in the whole hand after 12 weeks compared with baseline.Conclusion. No relationship was found between blood flow in the hands and the presence of DUs. After 12 weeks of bosentan treatment, blood flow had increased in the SSc patients but had not normalized to that of healthy subjects.Trial registration: https://www.clinicaltrialsregister.eu/ (EudraCT number 2010-023908-27). [ABSTRACT FROM PUBLISHER]

Objectives. Spleen tyrosine kinase (SYK) is a core signalling protein that drives inflammatory responses and is fundamental to the propagation of signals via numerous immune receptors, including the B cell receptor and Fc receptors (FcRs). Fostamatinib, a small molecule SYK inhibitor, has shown evidence of ameliorating inflammation in RA patients. We sought to understand how the active metabolite of fostamatinib, R406, affects the inflammatory response at the cellular level.Methods. Antigen-specific in vivo systems and in vitro fluorescence microscopy were combined to investigate the effects of fostamatinib on antigen-specific interactions between dendritic cells (DCs) and CD4+ T cells.Results. Although it has previously been shown that R406 reduces the response of DCs to immune complexes (ICs), we found that fostamatinib failed to reduce specific CD4+ T cell proliferation in mice after immunization with ICs. However, we observed in vitro that R406 reduces both the area and duration of cellular interactions between IC-activated DCs and specific CD4+ T cells during the initial phase of cellular crosstalk. This led to diminished proliferation of antigen-specific CD4+ T cells after R406 treatment compared with vehicle controls. This decreased proliferative capacity of CD4+ T cells was accompanied by reduced expression of the co-stimulatory molecules, inducible T cell co-stimulator (ICOS) and PD-1, and abrogation of the production of inflammatory cytokines such as IFN-γ and IL-17.Conclusion. Our findings indicate a potential mechanism by which this compound may be effective in inhibiting FcR-driven CD4+ T cell responses. [ABSTRACT FROM PUBLISHER]

Objective. Human umbilical cord Wharton jelly-derived mesenchymal stem cells (hUCMS) are easy to retrieve in bulk. They may interact with immune cells by either cell contact or soluble factors. Little evidence is currently available on potential therapeutic application of hUCMS to systemic autoimmune disorders such as primary SS (pSS). We have recently developed an endotoxin-free alginate gel that can be used to microencapsulate different cell types for graft into non-immunosuppressed hosts. We aimed to assess the in vitro effects of IFN-γ-pretreated microencapsulated (CpS)-hUCMS on T cells of pSS.Methods. Ten pSS patients and 10 healthy donors were selected. Peripheral blood mononuclear cells (PBMCs) were obtained from venous blood to establish co-cultures with CpS-hUCMS. Lymphocyte proliferation and phenotypic analysis was performed by flow cytometry and real-time PCR on IFN-γ-pretreated hUCMS was performed before PBMCs co-culture.Results. We found that CpS-hUCMS suppress pSS T cell proliferation and restore the Treg/Th17 ratio, thereby possibly positively impacting the pSS disease process.Conclusion. We have developed a new biohybrid drug delivery system that now waits for clinical application in autoimmune diseases, including pSS. [ABSTRACT FROM PUBLISHER]

Objectives. Nuclear factor κB (NF-κB) is a critical activator of inflammatory processes and MTX is one of the most commonly prescribed DMARDs for treatment of RA. We sought to determine whether MTX inhibited NF-κB activity in RA and in lymphocytes and fibroblast-like synoviocytes (FLSs) and to define underlying mechanisms of action.Methods. An NF-κB luciferase reporter plasmid was used to measure NF-κB activation across experimental stimuli. Flow cytometry was used to quantify changes in intracellular protein levels, measure levels of reactive oxygen species and determine apoptosis. Quantitative RT-PCR was used to identify changes in MTX target genes.Results. In T cell lines, MTX (0.1 μM) inhibited activation of NF-κB via depletion of tetrahydrobiopterin (BH4) and increased Jun-N-terminal kinase (JNK)-dependent p53 activity. Inhibitors of BH4 activity or synthesis also inhibited NF-κB activation and, similar to MTX, increased JNK, p53, p21 and JUN activity. Patients with RA expressed increased levels of phosphorylated or active RelA (p65) compared with controls. Levels of phosphorylated RelA were reduced in patients receiving low-dose MTX therapy. In contrast, inhibition of NF-κB activation by MTX was not mediated via BH4 depletion and JNK activation in FLSs, but rather was completely prevented by adenosine receptor antagonists.Conclusion. Our findings support a model whereby distinct pathways are activated by MTX in T cells and FLSs to inhibit NF-κB activation. [ABSTRACT FROM PUBLISHER]

Objective. The aim of this study was to test the possible involvement, relevance and significance of dentin matrix protein 1 (DMP1) in chondrocyte redifferentiation and OA.Methods. To examine the function of DMP1 in vitro, bone marrow stromal cells (BMSCs) and articular chondrocytes (ACs) were isolated and differentiated in micromasses in the presence or absence of DMP1 small interfering RNA and analysed for chondrogenic phenotype. The association of DMP1 expression with OA progression was analysed time dependently in the OA menisectomy rat model and in grade-specific OA human samples.Results. It was found that DMP1 was strongly related to chondrogenesis, which was evidenced by the strong expression of DMP1 in the 14.5-day mouse embryonic cartilage development stage and in femoral heads of post-natal days 0 and 4. In vitro chondrogenesis in BMSCs and ACs was accompanied by a gradual increase in DMP1 expression at both the gene and protein levels. In addition, knockdown of DMP1 expression led to decreased chondrocyte marker genes, such as COL2A1, ACAN and SOX9, and an increase in the expression of COL10A and MMP13 in ACs. Moreover, treatment with IL-1β, a well-known catabolic culprit of proteoglycan matrix loss, significantly reduced the expression of DMP1. Furthermore, we also observed the suppression of DMP1 protein in a grade-specific manner in knee joint samples from patients with OA. In the menisectomy-induced OA model, an increase in the Mankin score was accompanied by the gradual loss of DMP1 expression.Conclusion. Observations from this study suggest that DMP1 may play an important role in maintaining the chondrogenic phenotype and its possible involvement in altered cartilage matrix remodelling and degradation in disease conditions like OA. [ABSTRACT FROM PUBLISHER]

Objectives. We aimed to investigate whether CD147 can up-regulate the chemotactic, adhesive and invasive properties of human neutrophils and to determine the mechanism underlying this process.Methods. Human promyelocytic leukaemia cells (HL-60) cells and peripheral blood or synovial fluid neutrophils were isolated from RA patients. Under cyclophilin A (CypA) stimulation, chemotaxis, adhesion potential and invasion ability were assessed using chemotaxis, adhesion and invasiveness assays. Lipid raft isolation and western blot were used to determine the mechanism underlying the effects of CypA stimulation.Results. CD147 up-regulates the calcium-induced chemotaxis, adhesion ability and invasiveness of human neutrophils in RA patients. Transient receptor potential melastatin 7 may be responsible for this phenomenon.Conclusion. These findings suggest that in RA patients, abundant CypA up-regulates the calcium-induced chemotactic, adhesive and invasive properties of neutrophils via direct binding to CD147. Cyclophilin–CD147 interactions might contribute to the destruction of cartilage and bone in RA. [ABSTRACT FROM PUBLISHER]

Objective. The aim of this study was to investigate the cellular populations and regulatory factors responsible for B-lymphocyte stimulator (BLyS) overexpression in SLE patients.Methods. Surface and intracellular BLyS levels were quantified by flow cytometry in healthy and SLE monocytes cultured in the presence of TNF-α, IFN-α, IFN-γ, GM-CSF and SLE immune complexes (SLE-ICs), while soluble BLyS was measured by ELISA. Also, both surface and intracellular BLyS expression by different cell subsets was determined in 23 SLE patients and 16 healthy controls. Disease activity was assessed using classic BILAG index.Results. In vitro experiments using healthy monocytes showed that IFN-α and SLE-ICs induced a progressive increase in surface-bound BLyS with respect to the intracellular stores. IFN-α-treated SLE monocytes, especially from patients with high anti-dsDNA levels or disease activity, exhibited higher intracellular levels of BLyS that was mobilized to the membrane more rapidly and subsequently released. Furthermore, ex vivo analysis of SLE patients revealed up-regulated BLyS expression in B cells, myeloid and plasmacytoid dendritic cells (DCs), whereas active patients had an increased surface:intracellular BLyS ratio in monocytes and myeloid DCs.Conclusion. Monocyte BLyS induction and mobilization from intra- to extracellular compartments seems to be influenced by IFN-α and disease activity or anti-dsDNA levels. Accordingly, monocytes and myeloid DCs from active patients presented the highest membrane-bound:intracellular BLyS ratio. In addition, expression levels in several blood cells support the existence of generalized immune stimulation in SLE patients. [ABSTRACT FROM PUBLISHER]

Objective. Interstitial pneumonia (IP) is a chronic progressive interstitial lung disease associated with high mortality and poor prognosis. However, the pathogenesis of IP remains to be elucidated. The aim of this study was to clarify the role of CD161+ Vδ1+ γδ T cells in SSc patients with IP.Methods. The proportion of CD161+ Vδ1+ γδ T cells in peripheral blood mononuclear cells (PBMCs) and serum sialylated carbohydrate antigen (KL-6) levels were determined. GeneChip analysis was performed with CD161− and CD161+ Vδ1+ γδ T cells. Cytokine and chemokine expression from CD161+ Vδ1+ γδ T cells was measured and used to evaluate the effect of culture supernatant on fibroblast proliferation.Results. The proportion of CD161+ Vδ1+ γδ T cells was significantly higher in SSc than healthy controls (HCs) and correlated negatively with serum KL-6 levels in IP-positive SSc patients. The gene and mRNA expression level of chemokine ligand 3 (CCL3) was markedly higher in CD161+ Vδ1+ γδ T cells than in CD161− Vδ1+ γδ T cells. CD161+ Vδ1+ γδ T cells in IP-positive SSc patients showed higher production of CCL3 and lower production of IFN-γ than in HCs. Culture supernatant derived from IP-negative and IP-positive SSc patients promoted fibroblast proliferation, whereas that from HCs did not.Conclusion. The small proportion and the altered cell functions of CD161+ Vδ1+ γδ T cells among PBMCs in SSc patients play a role in the pathogenesis of IP. These findings suggest that CD161+ Vδ1+ γδ T cells may play a regulatory role in the pathogenesis of IP in SSc patients via IFN-γ production. [ABSTRACT FROM PUBLISHER]

Objective. The aim of this study was to investigate the effect of endogenous matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) on the invasive characteristics of RA synovial fibroblasts.Methods. Synovial fibroblasts isolated from patients with RA or OA were treated with MMP small interfering RNA (siRNA), inhibitors and recombinant proteins or TNF-α, with or without cartilage explants. Cell viability and proliferation were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and 5-bromo-2-deoxyuridine (BrdU) proliferation assays, respectively; apoptosis by an in situ cell death detection kit; migration and invasion by CytoSelect invasion assay, scratch migration and collagen gel assays; cartilage degradation by 1,9-dimethylmethylene blue assay; and inflammatory mediators and MMPs by ELISA, western blot and zymography.Results. MMP-2 was expressed by both OA and RA synovial fibroblasts, whereas only RA synovial fibroblasts expressed MMP-9. Suppressing MMP-2 or MMP-9 reduced RA synovial fibroblast proliferation equally. However, MMP-9 siRNA had greater effects compared with MMP-2 siRNA on promoting apoptosis and suppressing RA synovial fibroblast viability, migration and invasion. Suppression/inhibition of MMP-9 also decreased the production of IL-1β, IL-6, IL-8 and TNF-α, inactivated nuclear factor κB (NF-κB), extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) and suppressed RA synovial fibroblast-mediated cartilage degradation. In contrast, suppression/inhibition of MMP-2 stimulated TNF-α and IL-17 secretion and activated NF-κB, while recombinant MMP-2 (rMMP-2) inactivated NF-κB and suppressed RA synovial fibroblast-mediated cartilage degradation. Results using specific inhibitors and rMMPs provided supportive evidence for the siRNA results.Conclusion. Endogenous MMP-2 or MMP-9 contribute to RA synovial fibroblast survival, proliferation, migration and invasion, with MMP-9 having more potent effects. Additionally, MMP-9 stimulates RA synovial fibroblast-mediated inflammation and degradation of cartilage, whereas MMP-2 inhibits these parameters. Overall, our data indicate that MMP-9 derived from RA synovial fibroblasts may directly contribute to joint destruction in RA. [ABSTRACT FROM PUBLISHER]

Objective. The aim of this study was to clarify the mechanism of leucocytapheresis (LCAP) in patients with RA.Methods. Protein profiles of blood samples from two patients with RA obtained via LCAP column inlet and outlet lines were analysed by two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. The lactoferrin (LTF) levels of peripheral and circulating blood samples from seven patients obtained via the LCAP column blood circuit were then determined by ELISA. Peripheral blood samples from 14 patients with RA were exposed to unwoven polyester fibre filters and the LTF level was determined. In addition, morphological changes in neutrophils after exposure to the filter were examined by optical microscopy, electronic microscopy and LTF immunostaining.Results. LTF levels were increased in both samples from the LCAP column outlet and peripheral blood at the end of LCAP treatment. Furthermore, peripheral blood samples exposed to the filter revealed a decreased number of neutrophils and an increased level of LTF. Morphological analysis of the exposed neutrophils showed vacuolization of the cytoplasm and degranulation of LTF-positive granules. These data suggest that LTF stored in the granules of neutrophils is released from the neutrophils caught in the LCAP column.Conclusion. Because LTF has been reported to have multiple anti-inflammatory properties, increased levels of LTF may contribute to the clinical effect of LCAP in patients with RA. [ABSTRACT FROM PUBLISHER]

Objective. The B cell chemoattractant chemokine ligand 13 (CXCL13) is emerging as a new biochemical marker in RA. This study was undertaken to dissect the relationship between CXCL13 expression levels in the synovium and clinico-pathological variables relevant to RA pathogenesis and outcome.Methods. Synovial tissues from 71 RA patients were evaluated by immunohistochemistry. Thirty paired samples were used for comparative gene expression analysis by quantitative real-time PCR. CXCL13 levels were analysed in relation to cellular, molecular and clinical features of inflammation, lymphocyte activation and joint damage.Results. In patients with early disease (<12 months duration), CXCL13 expression correlated significantly with synovial markers of local disease activity and systemic inflammation. Such correlation was less evident in established RA. Notably, the association with lymphocyte infiltration and with expression of B/T cell–related activation and proliferation genes, such as activation-induced cytidine deaminase, IFN-γ and IL-2, remained highly significant independent of disease duration and local disease activity. Patients featuring the highest levels of CXCL13 were more frequently ACPA positive and IgG ACPA titres were increased in the high CXCL13 expression group. Furthermore, the frequency of erosive disease on radiographs was significantly higher in the upper tertile of CXCL13 expression (P = 0.01 with adjustment for disease duration and ACPA). Accordingly, synovial CXCL13 and the local receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG) ratio significantly co-varied (ρ = 0.52, P < 0.01), independent of the level of local inflammation.Conclusion. Synovial CXCL13 appears to be a marker of a more severe pattern of RA disease, characterized by increased lymphocyte activation and bone remodelling beyond the level of conventional markers of inflammation. [ABSTRACT FROM PUBLISHER]

Objective. CD70-expressing CD4 T cells are enriched in RA and promote autoimmunity via co-stimulatory CD70-CD27 interaction. This study aimed to explore the phenotype and cytokine production of CD70+ CD4 T cells in RA.Methods. Peripheral blood mononuclear cells from 32 RA patients were isolated and frequencies of CD70+ cells within different CD4 T subsets were analysed using flow cytometry. IFN-γ and IL-17 production were compared between the CD70+ and CD70− cells. Expression of master transcription factors T-bet, GATA3 and retinoic acid–related orphan receptor gamma t (RORγt) were examined by real-time PCR. Results are presented as mean (s.e.m.).Results. CD4 T cells of healthy controls rarely expressed CD70 as compared with CD4 T cells of RA patients [mean 0.9% (s.e.m. 0.3%) vs 7.6 (0.6), P < 0.001]. In RA, CD70+ cells were present within all CD4 T cell subsets, i.e. CD45RA+CCR7+ naive, CD45RA−CCR7+ central memory, CD45RA−CCR7− effector memory and CD45RA+CCR7− terminally differentiated effector memory T cells with a mean frequency of 3.9% (s.e.m. 1.1%), 4.0 (0.5), 4.2 (0.7) and 9.4 (4.3), respectively. As compared to CD70− CD4 T cells, CD70+ CD4 T cells produced significantly more IFN-γ and IL-17 after short activation. CD70+ CD4 T cells preferentially expressed transcription factor RORγt.Conclusion. CD70+ CD4 T cells are enriched in RA and may directly contribute to RA pathogenesis by producing IFN-γ and IL-17. Targeting CD70+ CD4 T cells might offer new therapeutic opportunities in RA. [ABSTRACT FROM PUBLISHER]

Objective. Regulatory B cells (Bregs) constitute a subset of B cells with immunomodulatory properties. Numerical and functional alterations in the Breg compartment have been associated with autoimmunity. The aim of this study was to assess the frequency and function of Bregs in patients with ANCA-associated vasculitis (AAV).Methods. B cell subsets were determined in the peripheral blood of 48 AAV patients (12 active, 36 in remission) and 41 healthy controls (HCs) by flow cytometry. Bregs were defined within the CD19+ population as CD24hiCD38hi or CD24hiCD27+ cells. The percentage of IL-10-positive B cells in circulation was analysed by flow cytometry. Sorted CD19+ B cells were co-cultured with monocytes to evaluate their capacity to inhibit monocyte TNF-α production upon lipopolysaccharide stimulation.Results. The frequency of circulating CD19+CD24hiCD38hi cells was not different in AAV patients in remission compared with HCs, but was decreased in patients with active disease [mean in HCs 5.5% (s.d. 1.6) vs active 3.8% (s.d. 2.8), P = 0.0104]. Furthermore, the percentage of CD19+CD24hiCD27+ cells was significantly decreased in both remission and active patients when compared with HCs [HCs 15.0% (s.d. 9.3) vs remission 6.6% (s.d. 4.4) (P < 0.0001) vs active 6.4% (s.d. 6.2) (P = 0.0006)]. The frequency of IL-10-positive B cells was comparable between patients and HCs. B cells from AAV patients suppressed monocyte TNF-α production to a similar extent to cells from HCs.Conclusion. Based on immunophenotypic classification, Bregs are numerically diminished in AAV patients. However, B cell function in terms of IL-10 production and their capacity to suppress monocyte activation is not compromised in AAV patients in remission. [ABSTRACT FROM AUTHOR]

Objectives. B cells are central to the pathology of ANCA-associated vasculitis (AAV), a disease characterized by autoantibodies and effectively treated by rituximab. In addition to promoting inflammation, a subset of B cells act to suppress harmful autoimmune responses (Breg). The balance of effector and regulatory B cell subsets in AAV is not known. This study was conducted to assess the relative frequency of these subsets during different states of disease activity.Methods. B memory (Bmem), naive (Bnaive) and regulatory (Breg) subsets were defined by their relative expression of CD24 and CD38. Function was assessed by cytokine production and suppressive action on CD4+ Th1 activation evaluated in a co-culture system.Results. Compared with healthy controls, the frequency of Breg (CD24hiCD38hi) was significantly reduced during disease remission in both proteinase 3 (PR3)- and MPO-ANCA patients and during acute disease in PR3-ANCA patients, while the frequency of memory cells (CD24hiCD38lo) was reduced during active disease and restored during remission. Breg cell frequency showed a positive correlation, while Bmem had an inverse correlation with IL-10 production in vitro. B and T cell co-cultures revealed that memory and naive B cell subsets augmented Th1 activation in vitro, which was prevented by Breg, and this pattern did not differ between remission AAV patients and controls.Conclusion. In remission there is a numerical, but not functional, deficiency in Breg and preservation of Bmem associated with reduced IL-10 production and increased Th1 activation in vitro. This imbalance may contribute to the high rate of relapse observed in AAV, especially in PR3-ANCA patients. [ABSTRACT FROM AUTHOR]

Biologic therapies that target pathogenic cytokines such as TNF, IL-1β or IL-6 have greatly improved the treatment of RA. Unfortunately, not all RA patients respond to current biologic therapies and responses are not always maintained, suggesting that there are alternative drivers of RA pathogenesis that might serve as promising therapeutic targets. Discovery of the new Th17 subset of Th cells, and their role in autoimmune disease development, has implicated the proinflammatory IL-12 and IL-17 families of cytokines in RA disease pathogenesis. Members of these cytokine families are elevated in the blood and joints of RA patients and have been shown to remain elevated in patients who do not respond to current biologics. In addition, these cytokines have been shown to play roles in joint destruction and erosion. A new subclass of biologics that target the IL-12 and/or IL-17 signalling pathways are under development. Here we review evidence for a role of Th17 cells as well as IL-12 and IL-17 cytokines in RA pathogenesis as the rationale for a subsequent discussion of the ongoing and completed clinical trials of newly emerging biologic therapies directed at IL-12 or IL-17 pathway inhibition. [ABSTRACT FROM AUTHOR]

Objective. The aim of this study was to evaluate the role of rituximab (RTX) in modulating the expression of the IL-17/IL-23 pathway in the salivary glands (SGs) of patients with primary SS (pSS).Methods. Consecutive SG biopsies were obtained from 15 patients with pSS before and after 1 year of RTX therapy. The SG expression of IL-17, IL-23p19 and p-STAT3 was evaluated by immunohistochemistry at baseline and after RTX therapy. The role of mast cells in pSS patients in modulating the Th17 response and the immunologic effect of RTX on mast cells were also studied in in vitro experiments.Results. IL-17 was overexpressed in the SGs of patients with pSS mainly by infiltrating T cells and mast cells. After RTX therapy, the SG expression of IL-17, but not of IL-23p19 and p-STAT3, was significantly reduced and was accompanied by the depletion of tissue mast cells. In in vitro experiments with heterologous peripheral lymphocytes RTX significantly induced the apoptosis of isolated mast cells. Finally, mast cells isolated from peripheral blood mononuclear cells of pSS patients in vitro significantly increased Th17 lymphocytes.Conclusion. RTX acts on pSS patients by globally reducing the expression of IL-17 and specifically inducing a pronounced apoptotic depletion of mast cells. [ABSTRACT FROM PUBLISHER]

Objective. The mechanisms of ectopic calcification in inflammatory diseases are poorly understood. We investigated the effects of inflammatory cytokines on the mechanisms of calcification in human adipose tissue-derived mesenchymal stem cells (hADSCs).Methods. The effects of inflammatory cytokines were evaluated using hADSCs cultured in osteoblast induction medium. mRNA expression was measured by real-time PCR and protein levels were measured by western blotting. Cell mineralization was evaluated by Alizarin Red S staining.Results. In hADSCs, administration of IL-6/soluble IL-6 receptor (sIL-6R), TNF or IL-1β accelerated calcification through enhanced expression of an osteoblast differentiation marker, runt-related transcription factor 2 (RUNX2). IL-6/sIL-6R had the greatest effect. The transcription of mRNA for receptor tyrosine kinase-like orphan receptor 2 (ROR2), involved in the non-canonical wingless-type (WNT) MMTV integration site pathway, was increased, while β-catenin expression, an essential factor in the canonical WNT signalling pathway for osteoblast differentiation, did not change. Suppression of signal transducer and activator of transcription 3 (STAT3), but not STAT1, by small interfering RNA (siRNA) exerted a strong inhibitory effect on RUNX2 and ROR2 expression, and inhibited accelerated calcification.Conclusion. IL-6/sIL-6R stimulation accelerated the ROR2/WNT5A pathway in hADSCs in a STAT3-dependent manner, resulting in augmented calcification. These results suggest that the mechanisms of ectopic calcification accelerated by IL-6 in hADSCs may be involved in chronic inflammatory tissues and that IL-6 inhibitors may be beneficial in the treatment of ectopic calcification in inflammatory diseases. [ABSTRACT FROM PUBLISHER]

Objective. Granulomatosis with polyangiitis (GPA) is a rare and sometimes fatal systemic autoimmune disease. ANCAs specific for PR3 are associated with GPA. Remission in GPA can be achieved through B cell depletion (BCD) therapy. Our aim was to understand whether the frequencies of T cell subsets are influenced by BCD.Methods. The frequencies of circulating T follicular helper cells (cTFHs) and regulatory T cells (Tregs) from 36 GPA patients including 11 rituximab-treated patients and 10 healthy controls were studied by flow cytometry. The functional capacity of Tregs was assessed by in vitro co-culture assays.Results. We observed an increased frequency of cTFHs and a reduced frequency of antigen-experienced Tregs in peripheral blood from GPA patients on conventional therapies but not in those treated with rituximab compared with healthy controls. Furthermore, the ratio of cTFHs to Tregs was significantly higher in GPA patients on conventional therapies than in GPA patients treated with rituximab who were clinically improved or controls. Whereas Tregs were numerically reduced in GPA patients on conventional therapy, the suppressive capacity of Tregs on a per cell basis was not significantly altered in these individuals.Conclusion. Our study illustrated increased cTFHs with decreased antigen-experienced Tregs in GPA patients on conventional therapies, but in B cell-depleted patients the levels of cTFHs and Tregs were similar to healthy controls. The negative correlation between cTFHs and Tregs implies the balance between T cell subsets and its B cell dependence impact on disease activity in GPA. [ABSTRACT FROM PUBLISHER]

Objective. The aim of this study was to identify serum markers that are modulated by an investigational anti-IFN-α mAb, sifalimumab, in adult DM or PM patients.Methods. In a phase 1b clinical trial, sera were collected from a total of 48 DM or PM adult patients receiving either placebo for 3 months or sifalimumab for 6 months. Samples were tested for 128 selected proteins using a multiplex luminex immunoassay. Muscle biopsies from selected patients were stained for T cell infiltration using an anti-CD3 antibody.Results. A robust overexpression of multiple serum proteins in DM or PM patients was observed, particularly in patients with an elevated baseline type I IFN gene signature in the blood or muscle. Neutralization of the type I IFN gene signature by sifalimumab resulted in coordinated suppression of T cell-related proteins such as soluble IL-2RA, TNF receptor 2 (TNFR2) and IL-18. Muscle biopsies from two patients with the highest serum protein suppression were selected and found to have a pronounced reduction of muscle T cell infiltration. Down-regulation of IL-2RA correlated with favourable manual muscle test 8 (MMT-8) alterations in sifalimumab-dosed patients.Conclusion. A reduced level of multiple T cell-associated proteins after sifalimumab but not placebo administration suggests a suppressive effect of blocking type I IFN signalling on T cell activation and chemoattraction that may lead to a reduction of T cell infiltration in the muscle of myositis patients. Further, soluble IL-2RA changes from baseline may serve as a responsive and/or predictive marker for type I IFN-targeted therapy in adult DM or PM patients. [ABSTRACT FROM PUBLISHER]

PM and DM are characterized clinically by weakness and low endurance of skeletal muscle. Other organs are frequently involved, suggesting that idiopathic inflammatory myopathies (IIMs) are systemic inflammatory diseases. Involvement of immune mechanisms in IIMs is supported by the presence of T cells, macrophages and dendritic cells in muscle tissue, by the presence of autoantibodies and by HLA-DR being a strong genetic risk factor. T cells may have direct and indirect toxic effects on muscle fibres, causing muscle fibre necrosis and muscle weakness, but the target of the immune reaction is not known. A newly identified T cell subset, CD28null T cells, may have cytotoxic effects in the CD4+ and CD8+ T cell phenotype. These cells are apoptosis resistant and may contribute to treatment resistance. Several myositis-specific autoantibodies have been identified, but they are all directed against ubiquitously expressed autoantigens and the specificity of the T cell reactivity is not known. These autoantibodies are associated with distinct clinical phenotypes and some with distinct molecular pathways; e.g. sera from patients with anti-Jo-1 autoantibodies may activate the type I IFN system and these sera also contain high levels of B cell activating factor compared with other IIM subsets. The characterization of patients into subgroups based on autoantibody profiles seems to be a promising way to learn more about the specificities of the immune reactions. Careful phenotyping of infiltrating immune cells in muscle tissue before and after specific therapies and relating the molecular findings to clinical outcome measures may be another way to improve knowledge on specific immune mechanism in IIMs. Such information will be important for the development of new therapies. [ABSTRACT FROM AUTHOR]

Objective. T helper 17 (Th17) and mast cells produce IL-17A in RA and critically contribute to the pathogenesis of RA. However, the complete IL-17 cytokine profile in RA is unknown. The aim of the study was to systematically study the expression of IL-17 family cytokines in RA.Methods. The expression of all IL-17 cytokines in RA synovium and pannus as well as in the synovium of OA was determined using quantitative RT-PCR (qRT-PCR). IL-17A and IL-17B were immunostained. Peripheral blood neutrophils were analysed for IL-17B. The effect of IL-17B alone or in combination with TNF-α was tested in vitro on fibroblasts and endothelial cells.Results. In all tissues IL-17B was the most expressed IL-17 family cytokine, found in lining but most strongly expressed in human neutrophil elastase containing polymorphonuclear cells. This pattern was distinct from that of IL-17A, which was found in mast cell tryptase immunoreactive cells. Circulating neutrophils contained IL-17B, verifying the in vivo results. Fibroblasts up-regulated the expression of IL-17RB, a putative receptor of IL-17B, after TNF-α stimulation. IL-17B significantly enhanced TNF-α-induced production of G-CSF and IL-6 in fibroblasts.Conclusion. IL-17B, which is present in synovium, may contribute to the pathogenesis of RA. IL-17B can enhance the effects of TNF-α on the production of cytokines and chemokines that control immune cell trafficking and neutrophil homeostasis in the inflamed tissues. [ABSTRACT FROM PUBLISHER]

Objectives. The objectives of this study were to characterize macrophages resident in inflamed articular adipose tissue (AAT) and non-inflamed subcutaneous adipose tissue (ScAT) of RA patients and to evaluate the basal and cytokine-triggered secretory activities of these tissues.Methods. Tissues were obtained from patients undergoing knee joint replacement surgery. The number of total CD68+, CD14+ and CD163+ macrophages was evaluated by immunohistochemistry. The concentrations of select factors were measured in supernatants from untreated and cytokine-treated tissue explant cultures using ELISA. IL-1β and TNF were applied as the stimuli.Results. Paired samples of AAT and ScAT, obtained from the same patients, contained a similar number of macrophages, displaying an M2-skewed phenotype. Both tissues released equivalent amounts of IL-1β, TNF, IL-10 and macrophage migration inhibitory factor (MIF). However, AAT secreted more chemokines (CCL2, CCL5), cytokines [IL-6, IL-8, IL-1 receptor antagonist (IL-1Ra)], hepatocyte growth factor (HGF) and MMP-3 than ScAT. Basal secretion of adipocytokines was not patient specific. Except for HGF and MIF, cytokine treatment up-regulated the release of these factors from both tissues, but also upon stimulation AAT produced more IL-6, IL-8 and IL-1Ra than ScAT.Conclusion. The secretory activity, reflecting cell activation status but not phenotype or the number of macrophages, discriminates rheumatoid AAT from ScAT. By releasing various factors possessing chemotactic, proinflammatory, anti-inflammatory and tissue degrading activities, AAT resident macrophages may drive and control local pathological processes. [ABSTRACT FROM PUBLISHER]

Objectives. Fibroblast-like synoviocytes (FLSs) are crucial players in the pathogenesis of synovitis in rheumatic diseases. Targeting FLS activation represents an approach to the development of therapeutic strategies. Our aim was to investigate whether the microenvironment of inflamed joints could modulate the expression of IL-22 and IL-22R1 on OA and RA FLSs. We also examined the effect of IL-22 on FLS activation as well as on their IL-17-related responses.Methods. IL-22 and IL-22R1 expression was studied by RT-PCR and immunoblotting. Proliferation was measured by an ELISA kit. IL-17 receptors, p19IL-23 and alarmins were analysed by RT-PCR. IL-17 receptor expression was evaluated by flow cytometry. MMP1 and IL-23 were measured by ELISA. S100A8/A9 expression was detected by immunofluorescence and ELISA. Signal transducer and activator of transcription 3 (STAT3) phosphorylation was quantified using a cell-based ELISA kit.Results. IL-22 and IL-22R1 were expressed constitutively in FLSs. We demonstrated that S100A8 and S100A9 were synthesized in FLSs. We reported that inflammatory mediators increased the expression of the IL-22/IL-22R1 axis, amplifying FLS activation. IL-22 enhanced FLS proliferation and up-regulated MMP1 and S100A8/A9 production. STAT3 phosphorylation was induced after IL-22 treatment and the stimulatory effect of IL-22 on S100A8/A9 was reduced after the activities of Janus kinase 2 (JAK2) and JAK3 were blocked. We showed an inhibitory action of IL-22 on IL-23 and IL-17RC expression in RA FLSs and on IL-17RA in OA FLSs.Conclusion. Therapies based on the pharmacological disruption signalling of IL-22 could be beneficial for the treatment of rheumatic diseases. The restricted expression of IL-22R1 to non-lymphoid cells could lead to a reduction of side effects mediated by immune responses. [ABSTRACT FROM PUBLISHER]

Objective. While systemic glucocorticoids compromise bone metabolism, altered intracellular cortisol availability may also contribute to the pathogenesis of primary male osteoporosis (MO). The objective of this study was to assess whether intracellular cortisol availability is increased in MO due to a distorted local cortisol metabolism.Methods. Forty-one patients with MO were compared with age- and BMI-matched non-osteoporotic subjects after excluding overt systemic hypercortisolism (N = 41). Cortisol, cortisone and the respective tetrahydro-, 5α-tetrahydro- and total cortisol metabolites were analysed by GC-MS in 24 h urine. Apparent 11β-hydroxysteroid dehydrogenase (11β-HSD) enzyme activities, excretion of cortisol metabolites and calcium, and fractional urinary calcium excretion were assessed and related to BMD.Results. Fractional and total urinary calcium excretion negatively correlated with BMD at all (P < 0.05) and at three of five (P < 0.05) measurement sites, respectively. While systemic cortisol was unchanged, apparent 11β-HSD enzyme activity in MO patients (P < 0.01) suggested increased intracellular cortisol availability. Total and fractional urinary calcium excretion was higher, with apparent 11β-HSD enzyme activities consistent with an enhanced intracellular cortisol availability (P < 0.05).Conclusion. Apparent 11β-HSD enzyme activities consistent with increased intracellular cortisol availability correlated with urinary calcium loss and reduced bone mineral density in MO. The changes in 11β-HSD activity were associated with both the fractional calcium excretion, suggesting altered renal calcium handling, and the absolute urinary calcium excretion. Both mechanisms could result in a marked bone calcium deficiency if insufficiently compensated for by intestinal calcium uptake. [ABSTRACT FROM PUBLISHER]